Myocardial infarction is one of the most widespread and serious health problems in Western society. In 2002, in the United States alone, over one million individuals suffered a myocardial infarction with over 25% fatality. During a heart attack, one or more of the arteries that supply the heart becomes blocked by a blood clot, usually at the site of fatty deposits known as arteriosclerosis. When victims are rushed to a hospital, blood flow is restored (the process known as reperfusion) either by drugs that dissolve clots or by angioplasty. Tissue salvage, however, is severely limited by free radicals and inflammatory responses, which cause as much as 80% of the damage during reperfusion (ischemia-reperfusion [I/R] injury). This, in turn, raises the risk of lethality and long-term complications.
A primary outcome of damage resulting from I/R injury is chronic congestive heart failure. Over 22% of male and 46% of female myocardial infarction victims will be disabled with congestive heart failure within six years following their heart attack. As the average age of the population increases and as survival following myocardial infarction improves, congestive heart failure will grow in importance. Following diagnosis of congestive heart failure, prognosis is poor. 12% of patients die within three months of diagnosis, 33% die within one year, and 60% die within five years.
Apart from accidental heart attacks, I/R injury is also a common outcome in cardiac surgery, leading to a spectrum of damage including arrhythmias, post-ischemic myocardial dysfunction, and cardiogenic shock. Furthermore, I/R injury is not limited to heart muscle, and also frequently occurs in the brain (stroke), liver, skeletal muscles and other organs.
Although the mechanism of I/R injury is not fully understood, a large body of evidence implicates a dual role for nitric oxide (NO) in this process. While NO is a potent cardioprotector, its excessive accumulation in ischemic tissue leads to the formation of reactive nitrogen species (e.g., peroxynitrite) that promote tissue injury by nitrating proteins. NO is a signaling molecule that is involved in a multitude of physiological processes including neurotransmission, immune regulation, vascular smooth muscle relaxation, and inhibition of platelet aggregation. See, e.g., Moncada S., Ann. N.Y. Acad. Sci. 1997; 811: 60-67; Ischiropoulos H., Arch Biochem Biophys 356: 1-11, 1998; Stamler et al., Cell 2001; 106: 675-683; Bian et al., J Pharmacol Sci. 2006; 101:271-9.
Depending upon the rate, timing, and spatial distribution of NO production, as well as the chemical microenvironment (e.g., presence of reactive oxygen species and redox status of the cell), NO acts either as a direct signaling messenger or as an indirect toxic effector via the formation of various reactive nitrogen species such as, e.g., peroxynitrite anion (ONOO−) and nitrogen dioxide (.NO2), formed as secondary products of .NO metabolism in the presence of oxidants including superoxide radicals (O2.−), hydrogen peroxide (H2O2), and transition metal centers. See Radi, Proc. Natl. Acad. Sci. USA, 2004, 101(12): 4003-4008.
NO is synthesized enzymatically from L-arginine by the enzyme nitric-oxide synthase (NOS) in almost all tissues of the body, including brain, peripheral nervous system, smooth muscle, kidney, vascular, lung, and uterus.
A large body of evidence has established the role of NO in the pathogenesis of inflammatory, infectious, and neurodegenerative diseases. The detrimental role of NO is rooted in the ability of its reactive metabolites to alter the function of biological macromolecules via covalent modifications of protein tyrosine, cysteine and tryptophane amino acid residues.
Cysteines and tryptophanes can be nitrosated to form S—NO and N—NO, respectively. These nitrosoderivatives are readily reversible (and form SH and NH2) in the presence of free thiols.
In contrast, tyrosine nitration has been considered to be an irreversible modification in vivo. Tyrosine nitration is mediated by reactive nitrogen species such as peroxynitrite anion (ONOO−) and nitrogen dioxide (.NO2). Once nitrated at tyrosine, proteins are thus thought to be irreparably damaged. Tyrosine nitration may affect protein structure and lead to loss of protein function or to a constitutively active proteins. For example, nitration of a tyrosine residue may prevent the subsequent phosphorylation of that residue. Alternatively, nitration of tyrosine residues may stimulate phosphorylation and result in constitutively active proteins. Furthermore, tyrosine nitration may change the rate of proteolytic degradation of nitrated proteins and favor either their faster clearance or accumulation in cells. See, e.g., Turko and Murad, Pharmacol. Reviews, 2002, 54(4): 619-634.
3-nitrotyrosine (3-NT) in body fluids and tissues has served as a biomarker of the involvement of NO in acute and chronic disorders such as I/R injury, atherosclerosis, diabetes, septic shock, Alzheimer's disease, Parkinson's disease, multiple sclerosis, pulmonary fibrosis, amyotrophic lateral sclerosis (ALS), inflammatory bowel disease, arthritis, allograft rejection, autoimmune myocarditis, pulmonary granulomatous inflammation, and cancer. Reviewed in, e.g., Ischiropoulos, Arch. Biochem. Biophys., 1998, 356(1): 1-11; Turko and Murad, Pharmacol. Reviews, 2002, 54(4): 619-634; Radi, Proc. Natl. Acad. Sci. USA, 2004, 101(12): 4003-4008. As previously observed by the present inventors and co-workers (see Rafikova et al., [Abstract] Nitric Oxide: Biology & Chemistry, 2006, 14:A67), tissue NO level is critical in the fate of cardiac tissue during I/R injury. Rats subjected to 30 minutes of myocardial ischemia (MI) and treated with sodium nitrite (5 mg/kg i.v.), infused 1 minute prior to ischemia set up, showed a significant expansion of I/R infarct size and myocardial tissue 3-NT accumulation compared with saline treated controls. In contrast, the use of NOS inhibitor L-NAME (50 mg/kg i.p.) provided the reduction in infarct size and 3-NT. It was also demonstrated that lowering tissue level of NO beyond certain point may also result in an enhanced myocardial damage. Thus, there exists an optimal tissue NO content that provides a minimal cell injury. Larger or smaller amounts of tissue NO are progressively more harmful probably due to either initiation of nitrosative stress or lack of NO antioxidant activity. For example, an excess of NO can lead to the formation of reactive nitrogen species, protein nitration, endothelial dysfunction, PARP and MMP activation, and mitochondrial respiration inhibition. These effects can lead to flow occlusion, apoptosis, necrosis, and inhibition of contractile function. However, a deficit of NO can also lead to detrimental effects since NO is a potent cardioprotector as it, e.g., induces cGMP synthesis, inhibits cytokine expression, and serves as a general antioxidant by intercepting oxygen radicals. As a result, NO in moderate amounts can improve perfusion, inhibit platelet aggregation, inhibit apoptosis, and increase ischemic tolerance.
The majority of current therapies for I/R injury, such as antiplatelet agents, anticoagulants, clot-dissolving drugs, vasodilators, and PTCA, target the occluded coronary artery rather than the ischemic tissue per se. Beta-blockers, which act by decreasing a tissue's O2 demand, are the only commercially available drugs that are protective against I/R damage.
Thus, there remains an unmet need in the art for safe and effective drugs that reduce the tissue damage associated with ischemia/reperfusion (I/R) injury of various tissues as well other types of tissue damage associated with septic shock and neurodegenerative diseases.